A drug delivery system should deliver drug at a rate dictated by the needs of a medical procedure over the period of the procedure, that is, the goal of any drug delivery system is to provide a therapeutic amount of drug to the proper site in the body to promptly achieve, and then maintain, the desired drug concentration. This objective emphasizes the need for spatial placement and temporal delivery of a drug or treatment. Spatial placement is the targeting of a drug to a specific organ, tissue, or bodily system such as the blood stream; while temporal delivery refers to controlling the rate of drug delivery to the target.
Targeted drug delivery systems include colloidal drug delivery systems and resealed or modified cells, for example, resealed or modified erythrocytes or leukocytes. Colloidal drug delivery systems include nanoparticles, microcapsules, nanocapsules, macromolecular complexes, polymeric beads, microspheres, liposomes, and lipid vesicles.
Liposomes are formed from phospholipids that are dispersed in an aqueous medium and spontaneously form multilamellar concentric bilayer vesicles (also termed multilamellar vesicles (MLVs)). MLVs generally have diameters of from 4 mm to 25 nm. Sonication or solvent dilution of MLVs results in the formation of small unilamellar vesicles (SUVs) with diameters in the range of 300 to 500 xc3x85.
Liposomes resemble cellular membranes, and water- or lipid-soluble substances can be entrapped in the aqueous spaces or within the bilayer, respectively. An important determinant in entrapping compounds is the physicochemical properties of the compound itself. Polar compounds are trapped in the aqueous spaces and are released through permeation or when the bilayer is broken; nonpolar compounds bind to the lipid bilayer of the vesicle, and tend to remain there unless the bilayer is disrupted by temperature or exposure to lipoproteins.
Liposomes may interact with cells via a number of different mechanisms, for example: endocytosis by phagocytic cells of the reticuloendothelial system such as macrophages and neutrophils; adsorption to the cell surface, either by nonspecific weak hydrophobic or electrostatic forces, or by specific interactions with cell-surface components; fusion with the plasma cell membrane by insertion of the lipid bilayer of the liposome into the plasma membrane, with simultaneous release of liposomal contents into the cytoplasm; or by transfer of liposomal lipids to cellular or subcellular membranes, or vice versa, without any association of the liposome contents. It often is difficult to determine which mechanism is operative and more than one may operate at the same time.
Intravenously injected liposomes may persist in tissues for hours or days, depending on their composition, and half-lives in the blood range from minutes to several hours. Larger liposomes are taken up rapidly by phagocytic cells of the reticuloendothelial system and exit only in places where large openings or pores exist in the capillary endothelium, such as the sinusoids of the liver or spleen. Thus, these organs are the predominant site of uptake. On the other hand, smaller liposomes show a broader tissue distribution but still are sequestered highly in the liver and spleen. In general, this in vivo behavior limits the potential targeting of liposomes to only those organs and tissues accessible to their large size. These include the blood, liver, spleen, bone marrow and lymphoid organs.
Attempts to overcome the limitation on targeting of liposomes have centered around two approaches. One is the use of antibodies, bound to the liposome surface, to direct the antibody and the liposome contents to specific antigenic receptors located on a particular cell-type surface. Further, carbohydrate determinants (glycoprotein or glycolipid cell-surface components that play a role in cell-cell recognition, interaction and adhesion) may also be used as recognition sites since they have potential in directing liposomes to particular cell types.
Further lipid vesicles, such as nonphospholipid paucilamellar lipid vesicles (PLV""s), are made from materials such as polyoxyethylene fatty esters, polyoxyethylene fatty acid ethers, diethanolamines, long-chain acyl amino acid amides, long-chain acyl amides, polyoxyethylene sorbitan mono and tristearates and oleates, polyoxyethylene glyceryl monostearates and monooleates, and glyceryl monostearates and monooleates, (U.S. Pat. Nos. 4,911,928, 4,917,951, and 5,000,960).
Resealed erythrocytes are another form of targeted drug delivery. When erythrocytes are suspended in a hypotonic medium, they swell to about one and a half times their normal size, and the membrane weakens, resulting in the formation of small pores. The pores allow equilibration of the intracellular and extracellular solutions. If the ionic strength of the medium then is adjusted to isotonicity, the pores will close and cause the membrane of the erythrocyte to return to normal or xe2x80x9cresealxe2x80x9d. Using this technique with a drug present in the extracellular solution, it is possible to entrap a substantial amount of the drug inside the resealed erythrocyte and to use this system for targeted delivery via intravenous injection.
Studies on the behavior of normal and modified reinfused erythrocytes indicate that, in general, normal aging erythrocytes, slightly damaged erythrocytes and those coated lightly with antibodies are sequestered in the spleen after intravenous reinfusion; but heavily damaged or modified erythrocytes are removed from the circulation by the liver. This suggests that resealed erythrocytes can be targeted selectively to either the liver or spleen, which can be viewed as a disadvantage in that other organs and tissues are inaccessible. Thus, the application of this system to targeted delivery has been limited mainly to treatment of lysosomal storage diseases and metal toxicity, where the site of drug action is in the reticuloendothelial system.
Labeling of red blood cells with chromium-51 and white blood cells with indium-111, as well as labeling of liposomes with contrast media and therapeutic agents is known. U.S. Pat. No. 5,466,438 relates to liposoluble complexes of paramagnetic ions and compounds bearing long acyl chains useful as magnetic resonance imaging contrast agents. U.S. Pat. No. 5,000,960 relates to coupling a molecule having a free sulfhydryl group to a lipid vesicle having a free sulfhydryl group incorporated as one of the structural molecules of the lipid phase thereby forming a covalent disulfide bond linkage. U.S. Pat. No. 4,931,276 relates to methods for introducing desired agents into red blood cells, and U.S. Pat. No. 4,478,824 relates to methods and apparatus for causing reversible intracellular hypertonicity in red blood cells of mammals in order to introduce desired materials into the cells, or achieve therapeutically desirable changes in the characteristics of intracellular hemoglobin. Further, poor accumulation of liposomal cadmium-texaphyrin in tumor tissue was cited as a possible explanation for low efficiency of photodynaric therapy in Kxc3x6nig et al., (Lasers in Surgery and Medicine 13:522, 1993; in: Photodynamic Therapy and Biomedical Lasers, P. Spinelli, M. Dal Fante and R. Marchesini, eds., Elsevier Science Publishers, 1992, 802).
Photodynamic therapy (PDT) is a treatment technique that uses a photosensitizing dye that produces cytotoxic materials, such as singlet oxygen (O2(1Dg)) from benign precursors (e.g. ((O2(3Sgxe2x80x94)), when irradiated in the presence of oxygen. Other reactive species such as superoxide, hydroperoxyl, or hydroxyl radicals may be involved. At the doses used, neither the light nor the drug has any independent activity against the disease target.
The effectiveness of PDT is predicated on three main factors: i) The photosensitive dyes used in PDT preferably have the ability to localize at the treatment site as opposed to surrounding tissue. ii) The high reactivity and short lifetime of activated oxygen means that it has a very short range and is unlikely to escape from the cell in which it is produced; cytotoxicity is therefore restricted to the precise region of photoactivated drug. iii) Developments in light delivery, such as lasers, light emitting diodes, and fiber optics, allow a beam of intense light to be delivered accurately to many parts of the body.
In recent years, considerable effort has been devoted to the synthesis and study of new photosensitizers (a review is found in Brown, S. B. and Truscott, T. G., 1993, Chemistry in Britain, 955-958). The development of more effective photochemotherapeutic agents requires the synthesis of compounds which absorb in the spectral region where living tissues are relatively transparent (i.e., 700-1000 nm), have high triplet quantum yields, are minimally toxic, and have physiologically acceptable water/lipid partition coefficients. Texaphyrins have proven to be effective sensitizers for generating singlet oxygen and for photodynamic therapy (U.S. Pat. Nos. 5,272,142; 5,292,414; 5,439,570; and 5,451,576, incorporated by reference herein).
Magnetic resonance imaging has become an important diagnostic tool in medicine, especially for tumor imaging. Imaging of tissue is dependent upon a difference in the relaxation rates of nuclear spins of water protons from various tissues in a magnetic field. The relaxation rate can be enhanced by use of a contrast agent, thereby improving a resulting image. The gadolinium cation is a superior contrast agent due to its seven unpaired f-electrons and high magnetic moment. However, gadolinium cation is too toxic to be used directly for imaging at concentrations required for effective enhancement. Texaphyrins bind the gadolinium ion in a stable manner and have proved to be nontoxic and effective contrast agents for imaging (U.S. Pat. Nos. 5,252,720, 5,451,576, and 5,256,399, incorporated by reference herein). Further development of texaphyrin-based magnetic resonance imaging protocols would be of significant value for the improvement of medical diagnostic imaging.
Macular degeneration due to damage or breakdown of the macula, underlying tissue, or adjacent tissue is the leading cause of decreased visual acuity and impairment of reading and fine xe2x80x9cclose-upxe2x80x9d vision. Age-related macular degeneration (ARMD) is the major cause of severe visual loss in the elderly. The most common form of macular degeneration is called xe2x80x9cdryxe2x80x9d or involutional macular degeneration and results from the thinning of vascular and other structural or nutritional tissues underlying the retina in the macular region. A more severe form is termed xe2x80x9cwetxe2x80x9d or exudative macular degeneration. In this form, blood vessels in the choroidal layer (a layer underneath the retina and providing nourishment to the retina) break through a thin protective layer between the two tissues. These blood vessels may grow abnormally directly beneath the retina in a rapid uncontrolled fashion; resulting in oozing, bleeding, or eyentually scar tissue formation in the macula which leads to severe loss of central vision. This process is termed choroidal neovascularization.
Neovascularization results in visual loss in other eye diseases including neovascular glaucoma, ocular histoplasmosis syndrome, myopia, diabetes, pterygium, and infectious and inflammatory diseases. In histoplasmosis syndrome, a series of events occur in the choroidal layer of the inside lining of the back of the eye resulting in localized inflammation of the choroid and consequent scarring with loss of function of the involved retina and production of a blind spot (scotoma). In some cases, the choroid layer is provoked to produce new blood vessels that are much more fragile than normal blood vessels. They have a tendency to bleed with additional scarring, and loss of function of the overlying retina. Diabetic retinopathy involves retinal rather than choroidal blood vessels resulting in hemorrhages, vascular irregularities, and whitish exudates. Retinal neovascularization may occur in the most severe forms.
Current diagnosis of ocular disorders often includes use of a fluorescein or indocyanine green angiogram. In this procedure, the dye is injected into the blood stream through a vein in the arm. Special filters are placed in the light path, and in front of the film, to permit only the fluorescent dye to be seen as it passes through the vessels in the retina Pictures of the vascular anatomy are taken of the retina and macula as the dye passes through the blood vessels of the back of the eye. Vascular occlusions or leakage of dye indicates abnormal vasculature. Optical coherence tomography is another technique that uses noncontact imaging and provides high-depth resolution in cross-sectional tomographs of the retina.
Current treatment of neovascularization relies on ablation of blood vessels using laser photocoagulation. However, such treatment requires thermal destruction of the tissue, and is accompanied by full-thickness retinal damage, as well as damage to medium and large choroidal vessels. Further, the patient is left with an atrophic scar and visual scotoma. Moreover, recurrences are common, and the prognosis for the patient""s condition is poor.
Developing strategies, such as PDT, have sought more selective closure of the blood vessels to preserve the overlying neurosensory retina PDT of conditions in the eye characterized by neovascularization has been attempted using the conventional porphyrin derivatives such as hematoporphyrin derivative and PHOTOFRIN(copyright) porfimer sodium. Problems have been encountered in this context due to interference from eye pigments. In addition, phthalocyanine and benzoporphyrin derivatives have been used in photodynamic treatment. PCT publication WO 95 24930 and Miller et al., (Archives of Ophthalmology, June, 1995) relate to treatment of eye conditions characterized by unwanted neovasculature comprising administering a green porphyrin to the neovasculature and irradiating the neovasculature with light having a wavelength of 550-695 nm. U.S. Pat. No. 5,166,197 relates to phthalocyanine derivatives reportedly useful for macular degeneration. Asrani and Zeimer (British Journal of Ophthalmology, 1995, 79:766-770) relate to photoocclusion of ocular vessels using a phthalocyanine encapsulated in heat-sensitive liposomes. Levy (Semin. Oncol. 1994, 21/6, suppl. 15 (4-10)) relates to photodynamic therapy and macular degeneration with porfimer sodium (PHOTOFRIN(copyright), requiring light of 630 nm and causing cutaneous photosensitivity that may last for up to 6 weeks), and benzoporphyrin derivative (BPD verteporfin, causing cutaneous photosensitivity of a few days). Lin et al. relate to the photodynamic occlusion of choroidal vessels using benzoporphyrin derivative BPD-MA. Further, BPD and tin purpurin (SnET2) are insoluble in aqueous solutions and require hydrophobic vehicles for administration.
Texaphyrins are aromatic pentadentate macrocyclic expanded porphyrinsxe2x80x9d useful as MRI contrast agents, as radiosensitizers and in photodynamic therapy. Texaphyrin is considered as being an aromatic benzannulene containing both 18- and 22-electron delocalization pathways. Texaphyrin molecules absorb strongly in the tissue-transparent 700-900 nm range, and they exhibit inherent selective uptake or biolocalization in certain tissues, particularly regions such as, for example, liver, atheroma or tumor tissue. Paramagnetic texaphyrins have exhibited significant tumor selectivity as detected by magnetic resonance imaging. Texaphyrins and water-soluble texaphyrins, method of preparation and various uses have been described in U.S. Pat. Nos. 4,935,498; 5,162,509; 5,252,720; 5,256,399; 5,272,142; 5,292,414; 5,369,101; 5,432,171; 5,439,570; 5,451,576; 5,457,183; 5,475,104 5,504,205; 5,525,325; 5,559,207; 5,565,552; 5,567,687; 5,569,759; 5,580,543; 5,583,220; 5,587,371; 5,587,463; 5,591,422; 5,594,136; 5,595,726; 5,599,923; 5,599,928; 5,601,802; 5,607,924; and 5,622,946; PCT publications WO 90/10633, 94/29316, 95/10307, 95/21845, and 96/09315; allowed U.S. patent application Ser. Nos. 08/484,551 and 08/624,311; and pending U.S. patent application Ser. Nos. 08/458,347; 08/657,947; 08/591,318; 08/700,277; and 08/763,451; each patent, publication, and application is incorporated herein by reference.
Problems with prior art drug and PDT delivery systems include lack of specificity, toxicity, expense, and technical difficulties, among others. Problems with prior art magnetic resonance imaging contrast agents include insufficient differential biolocalization, insufficient signal, toxicity, and slow clearance, among others. Because of these problems, known procedures are not completely satisfactory, and the present inventors have searched for improvements.
The present invention relates generally to the fields of targeted drug delivery, medical imaging, diagnosis, and treatment. More particularly, it concerns compositions having a texaphyrin-hpophilic molecule conjugate loaded into a biological vesicle; and methods for imaging, diagnosis and treatment using this loaded vesicle.
Accordingly, the present invention provides compositions comprising a texaphyrin-lipophilic molecule-vesicle complex. Such compositions include cells of the vascular system, such as red blood cells or white blood cells, and micellar vesicles such as liposomes or nonphospholipid vesicles, loaded with a texaphyrin conjugated to a lipophiliz molecule. When the texaphyrin portion of the complex is photosensitive and when the complex is irradiated, the complex ruptures, depositing its contents. The invention therefore includes methods for delivering diagnostic or therapeutic agents via loaded texaphyrin-lipophilic molecule-vesicle complexes.
xe2x80x9cLoadingxe2x80x9d means labeling of membranes of a vesicle, embedding into a vesicular membrane, or incorporation into the interior of a vesicle. In particular, loading would include attachment to or within cells circulating within the vascular system or to or within liposomes or other lipid vesicles.
A texaphyrin-lipophilic molecule-biological vesicle complex is an embodiment of the present invention. By xe2x80x9cbiological vesiclexe2x80x9d is meant a membranous structure having a lipid bilayer, or a micelle. By xe2x80x9clipid bilayerxe2x80x9d is meant a bimolecular sheet of phospholipids and/or glycolipids. A biological vesicle may be a cell, such as a red cell or white cell, or membranous fragment thereof; a liposomal membrane; a nonphospholipid vesicle, or a colloidal drug delivery system. In one embodiment of the present invention, the biological vesicle is a resealed red blood cell.
As used herein, a xe2x80x9clipophilic moleculexe2x80x9d is a molecule having a lipid-water distribution coefficient that is optimal for localization to lipid-rich tissues or materials compared to localization in surrounding nonlipid-rich tissues or materials. xe2x80x9cLipid-richxe2x80x9d means having a greater amount of triglyceride, cholesterol, fatty acids or the like. Lipophilic molecules that may be conjugated to a texaphyrin include cholesterol; steroids including progestagens such as progesterone, glucocorticoids such as cortisol, mineralocorticoids such as aldosterone, androgens such as testosterone and androstenedione, and estrogens such as estrone and estradiol; phospholipids such as phosphatidyl choline, phosphatidyl serine, phosphatidyl ethanolamine, phosphatidyl inositol, or cardiolipin; sphingolipids such as sphingomyelin; glycolipids such as cerebroside, or ganglioside; molecules having isoprenoid side chains such as vitamin K2, coenzyme Q10, chlorophyll, or carotenoids; low density lipoprotein (LDL); or the like. Preferred lipophilic molecules are steroids, more preferably estradiol, or cholesterol, for example.
A method for photodynamic therapy is also an aspect of the present invention. The method comprises administering a photosensitive texaphyrin-lipophilic molecule-vesicle complex to a subject, and irradiating the complex. Preferably, the vesicle portion of the complex is a red blood cell, and in one embodiment, the subject is a donor of the red blood cell.
When loaded with a photosensitive texaphyrin-lipophilic molecule conjugate, a loaded vesicle has utility as a diagnostic or therapeutic agent since the cell or liposome can be disrupted using an appropriate light source, thereby depositing a diagnostic or therapeutic agent in vivo. Therefore, a method for delivery of an agent to a targeted biological site is a further embodiment of the present invention. The method comprises i) loading a vesicle with a photosensitive texaphyrin-lipophilic molecule conjugate and the agent to form a complex; ii) allowing the complex to locate at the targeted biological site; and iii) irradiating the complex. The complex is lysed by irradiating, thereby delivering the agent to the targeted biological site. The agent may be a diagnostic agent, photodynamic therapy agent, a chemotherapeutic agent, a radiation sensitizing agent, or naturally occurring cellular contents of a cell. A preferred vesicle portion of a complex to be loaded is a red blood cell, a preferred lipophilic molecule portion of a complex is estradiol or cholesterol, and the photosensitive texaphyrin-lipophilic molecule conjugate may have a diamagnetic metal cation bound by the texaphyrin. A preferred diamagnetic metal cation is Lu(III), La(III), In(III), Y(III), Zn(II) or Cd(II); a most preferred diamagnetic metal cation is Lu(III). Availability of red blood cells loaded with a photosensitive texaphyrin-lipophilic molecule conjugate provides a method for delivering a therapeutic PDT agent to a desired site with a high concentration of oxygen. By presenting a PDT agent this way, it is expected that the patient will experience less toxicity.
The method of photolysis of loaded blood cells or liposomes involves at least two sources of specificity. A first source of specificity is the natural localization of loaded cells or liposomes into the blood, liver, spleen, bone marrow, or lymphoid organs. A second source of specificity is the positioning of the laser light. Such positioning of laser light, either by manual or mechanical means, would be particularly advantageous when the photolysis is to be effected at a particular biological locus, such as, for instance, a deepseated tumor site. Here, the fact that the-texaphyrins absorb light at wavelengths where bodily tissues are relatively transparent (700-900 nm) is particularly advantageous. This procedure allows for the effective implementation of light-based strategies at loci deep within the body with relatively little deleterious light-based photosensitization of other tissues where the texaphyrin conjugates are not localized or where the light is not focused.
Further, the present invention provides for the possibility of using the patient""s own blood for loading with a diagnostic or a therapeutic agent and a texaphyrin-lipophilic molecule conjugate. In so doing, a uniquely xe2x80x9ccustomizedxe2x80x9d therapy with reduced toxicity, increased circulation, and maximum therapeutic effect is provided.
Vesicles loaded with a photosensitive texaphyrin-lipophilic molecule conjugate and a chemotherapeutic drug have utility in conventional chemotherapy. In such a case, by directing laser light at a tumor and lysing the vesicle, a chemotherapeutic agent is released only in proximity to the cancer. In addition, a localized photodynamic therapeutic effect of irradiating the texaphyrin will occur.
Another embodiment of the present invention is a method of imaging. The method comprises the steps of administering a detectable texaphyrin-lipophilic molecule-vesicle complex to a subject, and imaging the complex.
When the detectable texaphyrin is fluorescent, imaging is by observing fluorescence of the texaphyrin. When the detectable texaphyrin is complexed with a paramagnetic metal cation, imaging is by magnetic resonance imaging. Further imaging methods include x-ray imaging, Raman scattering, magnetometry (bioluminescence), or gamma scanning when the texaphyrin is complexed with a gamma emitting isotope. For fluorescent imaging, texaphyrins may be activated by 400-500 nm light (the Soret band) or 700-900 nm light, preferably 700-800 nm, (the Q band) and, therefore, provide considerable versatility for use in humans.
The term xe2x80x9cfluorescentxe2x80x9d, as used herein, means that upon photoirradiation by light associated with the absorption profile of texaphyrin, light is emitted at a longer wavelength by the irradiated texaphyrin. All texaphyrins are fluorescent, albeit, to varying degrees, and texaphyrins complexed with Y(III), Lu(III), Gd(III), Dy(III), Eu(III), or Mn(III) are particularly preferred as fluorescent texaphyrins, for example.
In addition to fluorescent detection, texaphyrins may be imaged by x-radiation, by Raman scattering, or by magnetometry; further, texaphyrins complexed with a paramagnetic metal cation may be used for magnetic resonance imaging. Preferred paramagnetic metal cations for complexing with a texaphyrin include Mn(III), Mn(III), Fe(III), or trivalent lanthanide metals other than La(III), Lu(III), and Pm(III). More preferably, the paramagnetic metal is Mn(II), Mn(M), Dy(III), or Gd(III); most preferably, Gd(III). Any of various types of magnetic resonance imaging can be employed in the practice of the invention, including, for example, nuclear magnetic resonance (NMR), NMR spectroscopy, and electronic spin resonance (ESR). The preferred imaging technique is NMR.
Gamma particle detection may be used to image a texaphyrin complexed to a gamma-emitting metal. 51Chromium, 68gallium, 99technetium, or 111indium are preferred metals for complexing to texaphyrins for gamma particle scanning. Monochromatic X-ray photon sources may be used for imaging also.
The present invention is useful in imaging a patient generally, and/or in specifically diagnosing the presence of diseased tissue in a patient. The imaging process of the present invention may be carried out by administering a detectable texaphyrin-lipophilic molecule-vesicle complex of the invention to a patient, and then scanning the patient to obtain visible images of an internal region of a patient and/or of any diseased tissue in that region. The complexes of the present invention are particularly useful in providing images of the blood pool, liver, reticuloendothelial system, spleen, bone marrow, lymph nodes, and muscle; they are especially effective blood pool agents, and are highly effective at enhancing the liver and highly useful for improving the detection of hepatic metastases. Red blood cells loaded with a texaphyrin-lipophilic molecule conjugate, when injected intravenously, have been demonstrated to serve as a contrast agent for MRI Vesicles loaded with a paramagnetic texaphyrin-lipophilic molecule conjugate have utility as a blood pool contrast agent, facilitating the enhancement of normal tissues, magnetic resonance angiography, and marking areas of damaged endothelium by their egress through fenestrations or damaged portions of the blood vascular system. The patient may be any type of animal, but preferably is a mammal, and most preferably is a human.
Texaphyrin-lipophilic molecule conjugates and texaphyrin-lipophilic molecule-vesicle complexes are also provided for use in ocular diagnosis and therapy, in particular, therapy involving photodynamic therapy of conditions of the eye characterized by abnormal vasculature. Accordingly, an aspect of the present invention is directed to a method for carrying out angiography of the eye, i.e., observing, vasculature of an eye of a subjecl The method comprises the steps of administering a detectable texaphyrin-lipophilic molecule or texaphyrin-lipophilic molecule-vesicle complex to the subject; and observing the vasculature of the eye. Observing may be by fluorescence or other imaging methods as herein described.
In a further aspect of the invention, a method for treating an ocular condition of a subject characterized by abnormal vasculature is provided. The method comprises the steps of administering a photosensitive texaphyrin-lipophilic molecule conjugate or a photosensitive texaphyrin-lipophilic molecule-vesicle complex to the subject; and photoirradiating the vasculature. The method may further comprise the step of observing the ocular condition of the subject by imaging the texaphyrin as stated herein.
A method for photodynamic therapy of macular degeneration of a subject, comprising the steps of administering a photosensitive texaphyrin-lipophilic molecule conjugate or texaphyrin-lipophilic molecule-vesicle complex to the subject; and photoirradiating the macula is another aspect of the invention.
A method for observing and treating an ocular condition of a subject characterized by abnormal vasculature using a single agent is also an aspect of the invention. The method comprises the steps of administering a photosensitive fluorescent texaphyrin-lipophilic molecule or a photosensitive fluorescent texaphyrin-lipophilic molecule-vesicle complex to the subject; observing the ocular condition of the subject by fluorescence of the texaphyrin; and photoirradiating the vasculature.
For angiography, texaphyrins may be activated by 400-500 nm light (the Soret band) or 700-800 nm light (the Q band) and, therefore, provide considerable versatility for use in humans. For phototherapy, texaphyrins may be irradiated at 400-500 nm and at longer wavelengths of light where ocular tissues are relatively transparent, especially where light can penetrate blood and vascular tissue, i.e., 700-800 nm, especially at about 732 nm. Texaphyrins are particularly effective as visualizing agents in angiography of ocular blood vessels due to their localization in areas of abnormal permeability or damage as described in U.S. Ser. No. 08/763,451, incorporated by reference herein.
Texaphyrin-lipophilic molecules or texaphyrin-lipophilic molecule-vesicle complexes can be administered in a bolus injection allowing for a sufficiently large amount of drug to be present in the blood and for fast-turnaround between dosing and treatment. Further, texaphyrins are cleared quickly from the body; no toxicity to the eye has been observed in the use of texaphyrins in angiography.
A method of inducing formation of antibodies having binding specificity for a texaphyrin in a subject is also an aspect of the present invention. This method comprises administering a photosensitive texaphyrin-lipophilic molecule-vesicle complex to a subject, and irradiating the complex. Irradiating with light disrupts the vesicle, causing the contents to be deposited in the subject, thereby exposing the subject to the texaphyrin and inducing antibody production to texaphyrin. In this case, the texaphyrin may be considered a hapten; if the vesicle is a foreign cell, then the vesicle may be considered an adjuvant in addition to being the carrier that delivers the texaphyrin. By xe2x80x9cforeignxe2x80x9d is meant that the loaded vesicle is from a different species of animal than the animal into which the loaded cell is administered. For example, the cell for loading may be a goat cell, and the subject administered the loaded cell may be a rabbit.
In addition, a further immunogen may be loaded into the vesicle for inducing antibodies having binding specificity for that immunogen. Antibodies having binding specificity for the cellular contents of the disrupted cell may also be formed.
A further aspect of the invention is an antibody having binding specificity for a texaphyrin molecule. Such antibodies are useful for purification of a texaphyrin, for screening assays for the presence of a texaphyrin, or for the presence of texaphyrin degradation products from metabolic processes.
A method of making a texaphyrin-lipophilic molecule-cell complex is an aspect of the present invention. The method comprises i) obtaining a texaphyrin-lipophilic molecule conjugate, and ii) incubating a cell with the texaphyrin-lipophilic molecule conjugate in a hypotonic saline solution for a time and under conditions wherein a texaphyrin-lipophilic molecule-cell complex is formed An optional step is to include a drug or therapeutic agent when incubating in the hypotonic solution. A preferred cell is an erythrocyte. Advantages of using resealed or modified autologous erythrocytes as drug carriers include the fact that they are biodegradable, fully biocompatible, and nonimmunogenic; they exhibit flexibility in circulation time depending on their physicochemical properties; the entrapped drug is shielded from immunologic detection; and chemical modification of a drug is not required.
A method of making a texaphyrin-lipophilic molecule-liposome complex is an aspect of the present invention. The method comprises the step of incubating a texaphyrin-lipophilic molecule conjugate with a lipid or incorporating a texaphyrin-lipophilic molecule into a preformed liposome or micelle for a time and under conditions wherein a texaphyrin-lipophilic molecule-liposome complex is formed. An optional step is to include a drug or therapeutic agent during the incubation or incorporation.
In summary, a vesicle loaded with a texaphyrin-lipophilic molecule conjugate is useful in medical imaging, diagnosis, and therapy.
Following long-standing patent law convention, the terms xe2x80x9caxe2x80x9d and xe2x80x9canxe2x80x9d mean xe2x80x9cone or morexe2x80x9d when used in this application, including the claims.
Loading of a biological vesicle, such as a red blood cell (RBC), white blood cell (WBC), or a liposome with a texaphyrin-lipophilic molecule conjugate has previously not been shown. In the present invention, RBC""s were successfully loaded with GdT2BET-estradiol conjugate (GTE 1A). However, attempted loading with GdT2BET alone was not successful, thereby indicating that a lipophilic molecule xe2x80x9chandlexe2x80x9d is an important aspect of the texaphyrin conjugate for loading success. Although the examples that follow demonstrate loading of red blood cells, the invention is not limited thereto; it is contemplated that other cells may be loaded as well, such as stem cells, bone marrow cells, platelets, granulocytes, lymphocytes including T and B cells, monocytes, neutrophils, eosinophils, plasma cells, macrophage, dendritic cells, or a cell of mesenchymal, ectodermal, or endodermal origin. Macrophages loaded with a texaphyrin-lipophilic molecule conjugate are expected to have utility in the treatment of atheroma since macrophages complex with cholesterol to form foam cells, a component of early atheroma
Loaded vesicles will naturally biolocalize into the blood, liver, spleen, bone marrow or lymphoid organs. Due to the size of a vesicle, such as a red blood cell or a liposome, compared to the size of a texaphyrin-lipophilic molecule conjugate, it is expected that the vesicle will dominate in tenns of biolocalization, and any localizing effect of a site-directing lipophilic molecule or the inherent biolocalization of texaphyrins will be secondary. For example, a texaphyrin-estradiol conjugate loaded into a vesicle may have some specificity for an estradiol receptor if the estradiol is superficial to the vesicle. Similarly, a vesicle loaded with a texaphyrin-cholesterol conjugate may have localization to the liver in addition to the natural localization of the vesicle to the liver.
Human LDL is a physiologic serum protein metabolized by cells via uptake by high affinity receptors. In particular, neovascularization has been shown to have increased numbers of LDL receptors; and by increasing the partitioning of the texaphyrn into the lipoprotein phase of the blood, LDL is expected to more efficiently deliver texaphyrin to target tissue. A texaphyrin-LDL conjugate is selective for neovascularization since leakage of the conjugate is expected to occur only in neovasculature due to the large size of the conjugate. LDL can be isolated and purified according to the procedure of Hauel et al., (J. Clin. Invest., 34:1345, 1995).
In the loading of red blood cells of the present invention, red blood cells are separated from plasma and washed in normal saline. They are then treated with hypertonic saline which leaves them crenated with their internal salt concentration being higher than normal. The crenated cell pellet is resuspended in hypotonic saline containing a texaphyrin-lipophilic molecule conjugate. Because of the concentration difference between the cell interior and the hypotonic solution, water and the conjugate are driven into the cells. The cells are then washed several times in normal saline. This procedure results in a red blood cell with extensive labeling with the texaphyrin-lipophilic molecule conjugate. Further methods for loading cells are known to those of skill in this art in light of the present disclosure and may be utilized in the preparation of complexes of the present invention, for example, inducing an osmotic difference by use of sucrose solutions, treating with calcium chloride or calcium phosphate, or the like.
White cells are obtained from blood by, for example, centrifugation through Ficoll Hypaque media. This separates the white blood cells from plasma components and red blood cells. Other techniques for obtaining specific types of cells are known to one of skill in the art in light of the present disclosure.
Liposomes may be prepared by any number of techniques that include freeze-thaw, sonication, chelate dialysis, homogenization, solvent infusion, microemulsification, spontaneous formation, solvent vaporization, reverse phase, French pressure cell technique, or controlled detergent dialysis, for example. Such preparation methods are known to one of skill in the art in light of the present disclosure. Preparation may be carried out in a solution, such as a phosphate buffer solution, containing a texaphyrin-lipophilic molecule conjugate so that the conjugate is incorporated into the liposome membrane. Alternatively, the conjugate may be added to already formed liposomes. Liposomes employed in the present invention may be of any one of a variety of sizes, preferably less than about 100 nm in outside diameter, more preferably less than about 50 nm.
Micelles may be prepared by suspension of a texaphyrin-lipophilic molecule and lipid compound(s) in an organic solvent, evaporation of the solvent, resuspension in an aqueous medium, sonication and then centrifugation. Alternatively, the texaphyrin-lipophilic molecule may be added to preformed micelles, which micelles are made by methods known by one of skill in the art in light of the present disclosure.
Techniques and lipids for preparing liposomes and micelles are discussed in U.S. Pat. No. 5,466,438, and references cited therein. The disclosures of each of the foregoing references are incorporated herein by reference.
A texaphyrin-lipophilic molecule conjugate as used herein is an aromatic pentadentate expanded porphyrin analog with appended functional groups, at least one of which is a lipophilic molecule. Pendant groups may enhance solubility or biolocalization or may provide coupling sites for site-directing molecules.
Examples of texaphyrin-lipophilic molecule conjugates are those having structure I or structure II: 
M is H, or a divalent or trivalent metal cation. A preferred divalent metal cation is Ca(II), Mn(II), Co(II), Ni(II), Zn(II), Cd(II), Hg(II), Fe(II), Sm(III), or UO2(II). A preferred trivalent metal cation is Mn(III), Co(III), Ni(III), Fe(III), Ho(III), Ce(III), Y(III), In(III), Pr(III), Nd(III), Sm(III), Eu(III), Gd(III), Tb((III), Dy(III), Er(III), Tm(III), Yb(III), Lu(III), La(III), or U(III). Most preferred trivalent metal cations are Lu(III) and Gd(III).
R1-R4, R7 and R8 are independently hydrogen, halide, hydroxyl, alkyl, alkenyl, alkynyl, aryl, haloalkyl, nitro, formyl, acyl, hydroxyalkyl, alkoxy, hydroxyalkoxy, hydroxyalkenyl, hydroxyalkynyl, saccharide, carboxy, carboxyalkyl, carboxyamide, carboxyamidealkyl, amino, aminoalkyl, a lipophilic molecule, or a couple that is coupled to a lipophilic molecule.
R6 and R9 are independently selected from the groups of R1-R4, R7 and R8, with the proviso that the halide is other than iodide and the haloalkyl is other than iodoalkyl.
R5 and R10-R12 are independently hydrogen, alkyl, alkenyl, alkynyl, aryl, hydroxyalkyl, alkoxy, hydroxyalkoxy, hydroxyalkenyl, hydroxyalkynyl, carboxyalkyl, carboxyamide, carboxyamidealkyl, amino, aminoalkyl, or a couple that is coupled to a saccharide, or to a lipophilic molecule. The term xe2x80x9cnxe2x80x9d is an integer value less than or equal to 5.
R13 is alkyl, alkenyl, oxyalkyl, or hydroxyalkyl having up to about 3 carbon atoms and having rotational flexibility around a first-bound carbon atom. Rotational flexibility allows the rest of the group to be positioned outside the plane of the texaphyrin. Thus, for example, a preferred alkenyl is CH2xe2x80x94CHxe2x95x90CH2. The pyrrole nitrogen substituent is most preferably a methyl group. A texaphyrin having a methyl group attached to a ring nitrogen is described in U.S. Pat. No. 5,457,183, incorporated by reference herein.
In this texaphyrin-lipophilic molecule conjugate, at least one of R1-R12 is a lipophilic molecule or a couple that is coupled to a lipophilic molecule. In a more preferred embodiment, at least one of R1, R2, R3, R4, R7 and R9 is a lipophilic molecule, and more preferably is estradiol or cholesterol, or a couple that is coupled to estradiol or cholesterol. In a presently preferred embodiment, the texaphyrin-lipophilic molecule conjugate is the conjugate depicted herein as 1A or 1B.
Texaphyrins of the present conjugates may be metal-free or may be in a complex with a metal. Divalent and trivalent metal complexes of texaphyrins are by convention shown with a formal charge of n+, where n=1 or 2, respectively. The value xe2x80x9cnxe2x80x9d will typically be an integer less than or equal to 5; however, one skilled in the art in light of the present disclosure would realize that the value of n would be altered due to any charges present on substituents R1-R12.
It is understood by those skilled in the art that texaphyrin-metal complexes have one or more additional ligands providing charge neutralization and/or coordinative saturation to the metal ion. Such ligands include chloride, nitrate, acetate, cholate, and hydroxide, among others.
Photosensitive texaphyrins are used for PDT. A photosensitive texaphyrin may be a free-base texaphyrin or may be metallated with a diamagnetic metal. The term xe2x80x9cphotosensitive,xe2x80x9d as used herein, means that upon photoirradiation by light associated with the absorption profile of texaphyrin, texaphyrin effects the generation of oxygen products that are cytotoxic. Cytotoxic oxygen products may be singlet oxygen, hydroxyl radicals, superoxide, hydroperoxyl radicals, or the like. A photosensitive texaphyrin may be a texaphyrin metal complex, and in this embodiment, the metal M is a diamagnetic metal cation and the diamagnetic metal cation preferably is Lu(III), La(III), In(III), Y(III), Zn(II) or Cd(II). A more preferred diamagnetic metal cation is Lu(III).
Representative examples of alkanes useful as alkyl group substituents of the present invention include methane, ethane, straight-chain, branched or cyclic isomers of propane, butane, pentane, hexane, heptane, octane, nonane and decane, with methane, ethane and propane being preferred. Alkyl groups having up to about thirty, or up to about fifty carbon atoms are contemplated in the present invention. Representative examples of substituted alkyls include alkyls substituted by two or more functional groups as described herein.
Representative examples of alkenes useful as alkenyl group substituents include ethene, straight-chain, branched or cyclic isomers of propene, butene, pentene, hexene, heptene, octene, nonejne and decene, with ethene and propene being preferred. Alkenyl groups having up to about thirty or fifty carbon atoms, and up to about five double bonds, or more preferably, up to about three double bonds are contemplated in the present invention.
Representative examples of alkynes useful as alkynyl group substituents include ethyne, straight-chain, branched or cyclic isomers of propyne, butyne, pentyne, hexyne, heptyne, octyne, nonyne and decyne, with ethyne and propyne being preferred. Alkynyl groups having up to about thirty, or up to about fifty carbon atoms, and having up to about five or up to about three triple bonds are contemplated in the present invention.
The aryl may be a compound whose molecules have the ring structure characteristic of benzene, naphthalene, phenanthrene, anthracene, and the like, i.e., either the 6-carbon ring of benzene or the condensed 6-carbon rings of the other aromatic derivatives. For example, an aryl group may be phenyl or naphthyl, and the term as used herein includes both unsubstituted aryls and aryls substituted with one or more nitro, carboxy, sulfonic acid, hydroxy, oxyalkyl or halide substituents. In this case, the substituent on the phenyl or naphthyl may be added in a synthetic step after the condensation step which forms the macrocycle.
Among the halide substituents, chloride, bromide, fluoride and-iodide are contemplated in the practice of this invention with the exception of iodide for R6 and R9. R6 and R9 may have chloride, bromide or fluoride substituents. Representative examples of haloalkyls used in this invention include halides of methane, ethane, propane, butane, pentane, hexane, heptane, octane, nonane and decane, with halides, preferably chlorides or bromides, of methane, ethane and propane being preferred.
xe2x80x9cHydroxyalkylxe2x80x9d means alcohols of alkyl groups. Preferred are hydroxyalkyl groups having one to twenty, more preferably one to ten, hydroxyls. xe2x80x9cHydroxyalkylxe2x80x9d is meant to include glycols and polyglycols; diols of alkyls, with diols of C1-10 alkyls being preferred, and diols of C1-3 alkyls being more preferred; and polyethylene glycol, polypropylene glycol and polybutylene glycol as well as polyalkylene glycols containing combinations of ethylene, propylene and butylene.
Representative examples of oxyalkyls include the alkyl groups as herein described having ether linkages. xe2x80x9cOxyalkylxe2x80x9d is meant to include polyethers with one or more functional groups. The number of repeating oxyalkyls within a substituent may be up to 200, preferably is from 1-20, and more preferably, is 1-10, and most preferably is 1-5. A preferred oxyalkyl is O(CH2CH2O)xCH3 where x=1-100, preferably 1-10, and more preferably, 1-5.
xe2x80x9cOxyhydroxyalkylxe2x80x9d means alkyl groups having ether or ester linkages, hydroxyl groups, substituted hydroxyl groups, carboxyl groups, substituted carboxyl groups or the like.
Representative examples of thioalkyls include thiols of ethane, thiols of straigth-chain, branched or cyclic isomers of propane, butane, pentane, hexane, heptane, octane, nonane and decane, with thiols of ethane (ethanethiol, C2H5SH) or propane (propanethiol, C3H7SH) being preferred. Sulfate-substituted alkyls include alkyls as described above substituted by one or more sulfate groups, a representative example of which is diethyl sulfate ((C2H5)2SO4).
Representative examples of phosphates include phosphate or polyphosphate groups. Representative examples of phosphate-substituted alkyls include alkyls as described above substituted by one or more phosphate or polyphosphate groups. Representative examples of phosphonate-substituted alkyls include alkyls as described above substituted by one or more phosphonate groups.
Representative examples of caboxy groups include carboxylic acids of the alkyls described above as well as aryl carboxylic acids such as benzoic acid. Representative examples of carboxyamides include primary carboxyamides (CONH2), secondary (CONHRxe2x80x2) and tertiary (CONRxe2x80x2Rxe2x80x3) carboxyamides where each of Rxe2x80x2 and Rxe2x80x3 is a functional group as described herein.
Representative examples of useful amines include a primary, secondary or tertiary amine of an alkyl as described hereinabove.
xe2x80x9cCarboxyamidealkylxe2x80x9d means alkyl groups with secondary or tertiary amide linkages or the like. xe2x80x9cCarboxyalkylxe2x80x9d means alkyl groups having hydroxyl groups, carboxyl or amide substituted ethers, ester linkages, tertiary amide linkages removed from the ether or the like.
The term xe2x80x9csaccharidexe2x80x9d includes oxidized, reduced or substituted saccharide; hexoses such as D-glucose, D-mannose or D-galactose; pentoses such as D-ribose or D-arabinose; ketoses such as D-ribulose or D-fructose; disaccharides such as sucrose, lactose, or maltose; derivatives such as acetals, amines, and phosphorylated sugrs; oligosaccharides, as well as open chain forms of various sugars, and the like. Examples of amine-derivatized sugars are galactosamine, glucosamine, sialic acid and D-glucarine derivatives such as 1-amino-1-deoxysorbitol.
A couple may be described as a linker, i.e., the covalent product formed by reaction of a reactive group designed to attach covalently another molecule at a distance from the texaphyrin macrocycle. Exemplary linkers or couples are amides, amine, disulfide, thioether, ether, polyether, ester, or phosphate covalent bonds. PCT publication WO 94/29316 is incorporated by reference herein for providing syntheses of texaphyrin-conjugates having these types of linkages or couples.
In most preferred embodiments, conjugates and appended groups are covalently bonded to the texaphyrin via a carboncarbon, carbon-nitr6gen, carbon-sulfur, or a carbon-oxygen bond, more preferably a carbon-oxygen or a carbon-nitrogen bond.
In the practice of the present invention, preferred functionalizations for texaphyrin I or II are: when R6 and R9 are other than hydrogen, then R5 and R10 are hydrogen or methyl; and when R5 and R10 are other than hydrogen, then R6 and R9 are hydrogen, hydroxyl, or halide other than iodide. Other preferred functionalizations are where R6 and R9 are hydrogen, then R5, R10, R11, and R12 are independently hydrogen, phenyl, lower alkyl or lower hydroxyalkyl. The lower alkyl is preferably methyl or ethyl, more preferably methyl. The lower hydroxyalkyl is preferably of 1 to 6 carbons and 1 to 4 hydroxy groups, more preferably 3-hydroxypropyl. The phenyl may be substituted or unsubstituted.
In a presently preferred texaphyrin I or II, R1 is CH2CH3 or (CH2)2CH2OH; R2 and R3 are CH2CH3; R4 is CH3; R5, R6, and R9-R12 are H; R8 is a lipophilic molecule or a couple that is coupled to a lipophilic molecule; and R7 is H, OH, OCH3 or O(CH2CH2O)xCH3 where x is 1-10 and preferably 1-5, more preferably 3. Preferably, R8 is estradiol or cholesterol, or a couple that is coupled to estradiol or cholesterol.
A couple that is coupled to a lipophilic molecule may be further described as O(CH2CH2O)mxe2x80x94 where m is 1-10 and preferably 1-5, or as O(CH2)nCOxe2x80x94 where n is 1-10 and preferably 1-3.
Presently preferred texaphyrin-lipophilic molecule conjugates, T2BET-estradiol conjugates, are provided as 1A and 1B. 
xe2x80x9cT2xe2x80x9d refers to two hydroxyl groups on the tripyrrane portion of texaphyrin, xe2x80x9cBETxe2x80x9d refers to the ethoxy R groups on the benzene portion of the molecule, and estradiol is the lipophilic molecule of this conjugate. The synthesis of this conjugate is provided in Example 1.
In other presently preferred texaphyrin compounds I or II, R1-R12 are as in Tables A and B for texaphyrins A1-A108, and M is as defined hereinabove. While the cited texaphyrins are presently prefenrrd for use in the present invention, the invention is not limited thereto.
One skilled in the art of organic synthesis in light of the present disclosure and the disclosures in the patents, applications and publications incorporated by reference herein could extend and refine the referenced basic synthetic chemistry to produce texaphyrins having various substituents. For example, polyether-linked polyhydroxylated groups, saccharide substitutions in which the saccharide is appended via an acetal-like glycosidic linkage, an oligosaccharide or a polysaccharide may be similarly linked to a texaphyrin. A doubly carboxylated texaphyrin in which the carboxyl groups are linked to the texaphyrin core via aryl ethers or functionalized alkyl substituents could be converted to various esterified products wherein the ester linkages serve to append further hydroxyl-containing substituents. Polyhydroxylated texaphyrin derivatives may be synthesized via the use of secondary amide linkages. Saccharide moieties may be appended via amide bonds. Polyhydroxylated texaphyrin derivatives containing branched polyhydroxyl(polyol) subunits may be appended to the texaphyrin core via aryl ethers or ester linkages.
Treatment of carboxylated texaphyrins with thionyl chloride or p-nitrophenol acetate would generate activated acyl species suitable for attachment to monoclonal antibodies or other biomolecules of interest. Standard in situ coupling methods (e.g., 1,1xe2x80x2-carbonyldiimidazole) could be used to effect the conjugation.
Substituents at the R6 and R9 positions on the B (benzene ring) portion of the macrocycle are incorporated into the macrocycle by their attachment to ortho-phenylenediamine in the 3 and 6 positions of the molecule. Substituents at the R5 and R10 positions on the T (tripyrrane) portion of the macrocycle are incorporated by appropriate functionalization of carboxyl groups in the 5 positions of the tripyrrane at a synthetic step prior to condensation with a substituted ortho-phenylenediamine. A lipophilic molecule may be added after the condensation step to form the texaphyrin macrocycle.
Lipophilic molecules having an amine functionality are modified post-synthetically with an activated carboxylic ester derivative of a texaphyrin. In the presence of a Lewis acid such as FeBr3, a bromide-derivatized texaphyrin will react with an hydroxyl group of a lipophilic molecule to form an ether linkage between the texaphyrin linker and the lipophilic molecule. A couple that is coupled to a lipophilic molecule may be further described as O(CH2CH2O)mxe2x80x94 where m is 1-10 and preferably 1-5, or as O(CH2)nCOxe2x80x94 where n is 1-10 and preferably 1-3.
Texaphyrin-lipophilic molecule conjugates may be made by methods as described herein and as known and described in the art, such as in U.S. patents, in pending applications, previously incorporated by reference herein. Texaphyrins have a number of properties that lend themselves for use in imaging and photodynamic treatment protocols, for example: texaphyrins have inherent biolocalization, localizing to tumors, atheroma, or the liver; they have absorption in the physiologically important range of 700-900 nm; they provide stable chelation for an otherwise toxic metallic cation; and are sufficiently nontoxic for in vivo use.
An aspect of the present invention is use of texaphyrin-lipophilic molecules or texaphyrin-lipophilic molecule-vesicle complexes in ocular diagnosis and therapy; especially diagnostic angiograms, and photodynamic therapy of conditions of the eye characterized by abnormal vasculature. xe2x80x9cAbnormal vasculaturexe2x80x9d, as used herein, means undesirable vasculature; neovasculature; irregular, occluded, weeping, or inflamed ocular vessels or ocular tissues; inflammatory ocular membranes; abnormal conditions having to do with channeling of fluids in the ocular area, especially blood vessels; and includes conditions such as macular degeneration, glaucoma, disc or retinal neovascularization in diabetic retinopathy, pannus which is abnormal superficial vascularization of the cornea or conjunctiva, pterygium which is thickening of the bulbar conjunctiva on the cornea, conditions having retinal or choroidal neovasculature, ocular histoplasmosis syndrome, myopia, ocular inflammatory diseases, central serous retinopathy, subretinal neovascular membrane, or neovasculature induced by neoplasm, such as melanoma or retinal blastoma, for example.
xe2x80x9cObserving the vasculaturexe2x80x9d, as used herein, means carrying out an imaging procedure and collecting information from an angiogram where fluorescent texaphyrins are used, from an x-ray, or from magnetic resonance image, for example, to interpret the condition of the eye. The condition of the eye may be normal, or may include vascular leakage or occlusions, for example. As used herein, xe2x80x9ceyexe2x80x9d or xe2x80x9cocularxe2x80x9d includes the eye, underlying and adjacent tissue, and related tissues near and around the eye that have an influence on the functioning of the eye.
The parameters used for effective angiography and effective treatment in PDT methods of the invention are interrelated. Therefore, the dose is adjusted with respect to other parameters, for example, fluence, irradiance, duration of the light used in photodynamic therapy, and the time interval between administration of the dose and the therapeutic irradiation. Such parameters should be adjusted to produce significant damage to abnormal vascular tissue without significant damage to the surrounding tissue or, on the other hand, to enable the observation of blood vessels in the eye without significant damage to the surrounding tissue. Typically, the dose of texaphyrin of the texaphyrin-lipophilic molecule conjugate or texaphyrin-lipophilic molecule-vesicle complex used is within the range of from about 0.1 to about 50 xcexcmol/kg/treatment, and preferably from about 0.10-20 xcexcmol/kg/treatment. Further, as the texaphyrin dose is reduced, the fluence required to treat neovascular tissue may change.
After the photosensitizing texaphyrin-lipophilic molecule conjugate or texaphyrin-lipophilic molecule-vesicle complex has been administered, the tissue being treated in the eye is irradiated at the wavelength of maximum absorbance of the texaphyrin, usually either about 400-500 nm or about 700-800 nm. The light source may be a laser, a light-emitting diode, or filtered light from, for example, a xenon lamp; the light may have a wavelength range of about 400-900 nm, preferably about 400-500 nm or 700-800 nm, more preferably about 730-770 nm; and the light may be administered topically, endoscopically, or interstitially (via, e.g., a fiber optic probe). Preferably, the light is administered using a slit-lamp delivery system. A wavelength in this range is especially preferred since blood and retinal pigment epithelium are relatively transparent at longer wavelengths and, therefore, treatment results in less tissue damage and better light penetration. The fluence and irradiance during the irradiating treatment can vary depending on type of tissue, depth of target tissue, and the amount of overlying fluid or blood.
The optimum length of time following texaphyrin-lipophilic molecule conjugate or texaphyrin-lipophilic molecule-vesicle complex administration until light treatment can vary depending on the mode of administration, the form of administration, and the type of target tissue. For example, a time interval of minutes to about 5 h should be appropriate for vascular tissue. The time of light irradiation after administration may be important as one way of maximizing the selectivity of the treatment, thus minimizing damage to structures other than the target tissues. For a human, it is believed that the texaphyrin-lipophilic molecule conjugate or texaphyrin-lipophilic molecule-vesicle complex begins to reach the retinal and choroidal vasculature within seconds following administration, and persists for a period of minutes to hours, depending on the dose given. Treatment within the first five minutes following administration should generally be activated with focused light. At later time points, both focused or general illumination may be used.
In addition, texaphyrin-lipophilic molecule conjugate or texaphyrin-lipophilic molecule-vesicle complex can be used to observe the condition of blood vessels as a single agent, or in concert with other dyes such as fluorescein or indocyanine green to follow the progress of destruction of abnormal vascular tissue. In such angiographic systems, a sufficient amount of texaphyrin-lipophilic molecule conjugate or texaphyrin-lipophilic molecule-vesicle complex is administered to produce an observable fluorescent emission when excited by light, preferably light having a wavelength in the range of about 430-480 nm. Images are recorded by illuminating the eye with light in the excitation wavelength range and detecting the amount of fluorescent light emitted at the emission wavelength of about 730-760 nm. A preferred device, which both emits and receives light in the 430-760 nm range, is the TOPCON(trademark) 50VT camera in the Ophthalmic Imaging System (Ophthalmic Imaging System Inc., 221 Lathrop Way, Suite 1, Sacramento Calif.).
A camera is used to collect the emitted fluorescent light, digitize the data, and store it for later depiction on a video screen, as a hard paper copy, or in connection with some other imaging system. While a film recording device may be used when additional dyes such as fluorescein are being used in combination with the texaphyrin-lipophilic molecule conjugate or texaphyrin-lipophilic molecule-vesicle complex, a CCD camera (charge-coupled device) is preferable as being able to capture emissions at higher wavelengths. As a result, one can obtain more sophisticated information regarding the pattern and extent of vascular structures in different ocular tissue layers, giving the ability to detect the xe2x80x9cleakinessxe2x80x9d that is characteristic of new or inflamed blood vessels. Further, it is preferable to use a camera that is capable of providing the excitation light, appropriately filtered to deliver only light of the desired excitation wavelength range, and then to capture the emitted, fluorescent light with a receiving device, appropriately filtered to receive only light in the desired emission wavelength range.
For the above-described uses, texaphyrin-lipophilic molecule-cell or -liposome complexes are provided as pharmaceutical preparations. A pharmaceutical preparation of such a complex may be administered alone or in combination with pharmaceutically acceptable carriers, in either single or multiple doses. Suitable pharmaceutical carriers include inert solid diluents or fillers, sterile aqueous solution and various organic solvents. The pharmaceutical compositions formed by combining a complex of the present invention and the pharmaceutically acceptable carriers are then easily administered in a variety of dosage forms such as injectable solutions.
For parenteral administration, suspensions of the liposomal complex in sesame or peanut oil, aqueous propylene glycol, or in sterile aqueous solution may be employed. Such aqueous solutions should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration. In this connection, sterile aqueous media which can be employed will be known to those of skill in the art in light of the present disclosure. Intravenous administration of loaded red or white blood cell complexes of the present invention is contemplated as the most preferred method of administration.
Sterile technique is used for removal of cells from a patient, loading with a sterile texaphyrin-lipophilic molecule conjugate and replacement of loaded cells into the same patient. A pharmaceutically acceptable carrier may be used, which carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars such as mannitol or dextrose or sodium chloride. A more preferable isotonic agent is a mannitol solution of about 2-8% concentration, and, most preferably, of about 5% concentration.
Sterile conjugate solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
As used herein, xe2x80x9cpharmaceutically acceptable carrierxe2x80x9d includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic compositions is contemplated. Supplementary active ingredients can also be incorporated into the compositions.
For fluorescent detection methods of the present invention, a sufficient amount of texaphyrin is administered to produce an observable fluorescent emission when excited by light, preferably light having a wavelength in the range of about 430-480 nm. Images are recorded by illuminating with light in the excitation wavelength range and detecting the amount of fluorescent light emitted at the emission wavelength of preferably about 730-760 nm. Such dose can be determined without undue experimentation by methods known in the art or as described herein.
The complexes to be used in the photodynamic methods of the present invention are administered in a pharmaceutically effective amount. By xe2x80x9cpharmaceutically effectivexe2x80x9d is meant that dose which will, upon exposure to light, cause disruption of the loaded vesticle. The specific dose will vary depending on the particular complex chosen, the dosing regimen to be followed, photoirradiation exposure, and timing of administration. Such dose can be determined without undue experimentation by methods known in the art or as described herein.
The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventors to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.